Improved KIR gene and HLA-C KIR ligand sequence-specific primer polymerase chain reaction genotyping using whole genome amplification.

Molecular analysis of genetic polymorphism for clinical or research purposes may be compromised by genomic DNA of limited quality and quantity. In this study, we have successfully tested the feasibility of using whole genome amplification (WGA) to allow genotyping for killer cell immunoglobulin-like receptor (KIR) genes and human leucocyte antigen (HLA)-C KIR ligand dimorphism on HLA-C. WGA was achieved by multiple displacement amplification (MDA) using bacteriophage phi29 polymerase. For KIR genotyping, a revised sequence-specific primer polymerase chain reaction protocol consisting of 23 primer pairs was used avoiding hitherto undetected cross-priming involving KIR2DL1, KIR2DS1, KIR3DL1 and KIR3DS1 alleles. Similarly, MDA-amplified genomic DNA was analyzed for the detection of the HLA-C KIR ligand groups C1 and C2, based on the amino acid K/N dimorphism in position 80.

MICA compatibility and immunization in third kidney transplantations.

Significantly lower graft survival has been observed among recipients of a third (G3) compared with a first or second kidney transplantation. Because patients awaiting G3 are largely HLA immunized, they are usually transplanted with a high HLA match. Moreover, their rate of acute rejection episodes is similar to a first or second transplantation. Since major histocompatibility complex class I related chain A (MICA) molecules have been proposed as new targets for antibody recognition, we were interested to type donors and recipients for MICA alleles and to study MICA immunization of these patients. Forty-three pairs of donors and recipients were typed for MICA alleles using Luminex technology (LABtype RSSO). MICA alleles showed strong linkage disequilibrium with the B locus: some 4-digit alleles were preferentially associated with a given MICA allele. A greater frequency of patients with 2 MICA mismatches (MM) was observed among patients with rejection (40%), whereas all the graft losses were observed in patients with 0 or 1 MICA MM. MICA immunization was studied using sera from 52 patients collected on day 0 and after transplantation using a Luminex assay (LABScreen). MICA immunization was less frequent than HLA immunization, and MICA donor-specific antibody (DSA) was equally present in functional and failed grafts. These observations confirmed the potential role of MICA immunization in rejection, whereas the poor graft survival among third transplantations could not be explained by MICA incompatibility or immunization.

Specificity of T cells invading the skin during acute graft-vs.-host disease after semiallogeneic bone marrow transplantation.

The mechanisms responsible for skin lesions during acute graft-vs.-host disease (aGVHD) after allogeneic bone marrow transplantation (BMT) are poorly understood. The exact role of various effector cell populations and "major" (particularly HLA-DP) or "minor" antigens as target molecules is not known. To investigate the nature of cells responsible for tissue injury, we cultured T cells from skin biopsy first with interleukin 2 (IL-2) alone and then in polyclonal activation conditions to avoid in vitro antigenic sensitization before specificity testing. We applied this method to two biopsies performed during aGVHD after semiallogeneic BMT and obtained cytotoxic T cells against four graft mismatches: CD8+ T cells against HLA-A2.2 and HLA-B27 and CD4+ T cells against HLA-DP101 and HLA-DP401. This demonstrates that T cells with documented specificity can be obtained from an aGVHD lesion without antigenic selection. Moreover, these data directly implicate DP as a potential target antigen for aGVHD.

[Post-transfusion acute lung injury (Trali) after plasma infusion in a patient having a constitutional thrombotic microangiopathy]. / Syndrome d'oedème pulmonaire lésionnel aigu post-transfusionnel (Trali) après perfusion de plasma frais congelé au cours d'une microangiopathie thrombotique congénitale.

INTRODUCTION: Transfusion-related acute lung injury is a post-transfusion interstitial lung injury. CASE REPORT: We reported a post-transfusion acute lung injury in a 23-years old woman having a chronic thrombotic microangiopathy related to an ADAMTS 13 constitutional deficiency receiving monthly plasma infusion for six years. The temporal relationship between the lung injury and the infusion of fresh frozen plasma led to the diagnosis of transfusion-related acute lung injury. The finding in the donor of the transfused plasma of an anti-HLA class II antibody recognizing HLA-DR52 present on leucocytes of the recipient suggests a causal relationship between this antigen-antibody conflict and the triggering of the TRALI. This chronic pathologic state requiring monthly plasma transfusions for thrombotic accident prevention raises the question of the selection of plasma obtained from non-immunized donors. CONCLUSION: The occurrence of a post transfusion pulmonary edema without cardio-vascular overload, must lead to consider a TRALI especially in predisposing clinical situations. In the case reported the role of constitutional ADAMTS 13 deficiency in genesis of TRALI is considered.

Preparative two-step purification of human IL-2 by HPLC and hydrophobic affinity chromatography.

Interleukin 2 (IL-2) has been purified by a protocol using gel filtration high performance liquid chromatography (HPLC) and hydrophobic affinity chromatography with blue-trisacryl M. Peripheral blood lymphocytes or tonsillar lymphocytes were stimulated with phytohemagglutinin (PHA). Serum free conditioned medium (CM) containing IL-2, other lymphokines and residual PHA molecules was analyzed after 3 variations of ammonium sulfate (AS) precipitation: (1) precipitation of CM with 50% AS yielded a precipitate containing most of the residual PHA but also a fraction of IL-2. (2) Precipitation with direct 80% AS of crude CM yielded both IL-2 and residual PHA. (3) A double step procedure (50% AS followed by 80% AS) yielded a precipitate containing IL-2 but free of residual lectin. HPLC purification of these various AS-precipitated materials or of lyophilized crude CM yielded 2 peaks with mitogenic activity as assayed with the CTLL2 murine clone or IL-2-dependent human Con A-stimulated lymphoblasts. IFN was easily separated from IL-2 and PHA, but BCGF still copurified with IL-2. Peak I (25 kDa) was enriched 400-fold for IL-2 while peak II (68 kDa) contained the residual PHA. The IL-2-containing fractions eluted from HPLC were further purified by blue-trisacryl M chromatography. The IL-2 eluted with 0.4 M NaCl. The entire protocol (HPLC followed by blue-trisacryl) led routinely to 8000-fold IL-2 enrichment. Preparative HPLC directly applied to lyophilized crude (CM) enriched IL-2 activity 400-fold with yield averaging 60% of the IL-2 input. The final material was free from interferon and IL-1, but BCGF still copurified with IL-2. The 2-step purified material (HPLC and blue-trisacryl) gave 2 bands in SDS-PAGE both of which contained IL-2.

The influence of HLA A-B-DR matching on cytomegalovirus disease after renal transplantation. Evidence that HLA-DR7-matched recipients are more susceptible to cytomegalovirus disease.

We examined the prevalence of cytomegalovirus infectious episodes, as defined by clinical, virological, and serological criteria (i.e., CMV disease), in 660 kidney graft recipients; 109 patients (16.5%) developed the disease, and 551 did not. No significant statistical link between CMV disease prevalence and a given HLA-A, -B, or -DR allele was observed. However, patients with HLA-DR7 matched grafts were statistically more frequently found (P < 0.01) in the group of recipients who developed CMV disease as compared with the group who did not develop CMV disease. Furthermore, among patients who developed CMV disease, a significant increase of HLA-DR7 matched over DR7 mismatched patients was noted, whereas no difference between matched and mismatched recipients for the other HLA-DR alleles was found. No difference in the severity of graft failure, often observed during, or immediately after, the CMV episode, was noted between patients matched or mismatched for HLA-DR7. Our data suggest that donor/recipient matching for HLA-DR7 is associated with increased CMV disease.

Systematic transfusion in hemodialyzed patients awaiting grafts: kinetics of anti-t and b lymphocyte immunization and its incidence on graft function.

Since June 1977, a systematic blood transfusion (BT) policy (160 ml of leukocyte-poor washed erythrocytes given every 6 months) has been applied to 126 hemodialyzed patients awaiting a first kidney graft. Only patients who had anti-T or B lymphocyte (T or BLY) antibodies (Ab) killing fewer than 10 or 20% of the panel cells, respectively, entered the protocol. Screening of anti-T and BLY was performed 8, 15, and 21 days after each BT. Patients were removed from the protocol if they developed Ab against more than 10 or 20% of the T or BLY panel cells. The cumulative immunization (all Ab types) averaged 90% after four BTs. Anti-BLY (63%) were more frequent than anti-TLY (49%) after for BTs. No anti-HLA-DR specificity could be attributed to the anti-BLY, whereas 20% of the anti-TLY displayed a particular anti-HLA-A,B specificity. Patients that had had BTs or pregnancies before entering the protocol had a higher degree of immunization. The kinetics of the anti-B or TLY pattern differed greatly both at the level of their detection after 8, 15, and 21 days following one BT and in their development after repeated BTs. Forty-three patients received transplants at various stages of the protocol. Recipients grafted without Ab had the best graft outcome (87 versus 66 actuarial percentage at 3 months), even though their HLA (A,B and DR) matching was inferior. There was no significant difference in recipients who had different subgroups of Ab. These data indicate that immunization is very high even after a few BTs when careful controls are performed and they suggest that BTs do not act via active enhancement.

Effect of protein A immunoadsorption on panel lymphocyte reactivity in hyperimmunized patients awaiting a kidney graft.

Six hyperimmunized patients awaiting a kidney graft underwent immunoadsorption of separated plasma through a protein-A column to remove HLA antibodies. This procedure was partially limited by constant and rapid antibody resynthesis in spite of strong immunosuppression with cyclophosphamide and prednisolone. Only two patients could be grafted with previously positive--currently negative crossmatches. The first died of infection on day 40, having developed early chronic vascular rejection and the other returned to hemodialysis on day 285 after the development of transplant glomerulopathy. However, immunoadsorption seems to be effective in removing HLA antibodies having a titer below 1/2. Such extremely hyperimmunized patients should probably be excluded from the immunoadsorption program.

Late acute failure of well-HLA-matched renal allografts with capillary congestion and arteriolar thrombi.

Seventeen cases of a histologically and clinically unusual renal acute dysfunction in kidney recipients, individualized among a population of 1378, are reported. The basic histological lesion was a huge capillary congestion, associated with capillary and arteriolar thromboses or parenchymal necrosis in most patients, and contrasting with the absence of the classical features of acute cellular rejection, i.e., tubulitis, glomerulitis, edema, and infiltrate. The corresponding clinical history was characterized by its early timing in the course of transplantation (< 3 months), its sudden occurrence in patients usually having good transplant function, leading to end-stage renal failure in a few days, and its resolution under rejection treatment. The occurrence of this syndrome was significantly linked with a good HLA matching: 13 of the 17 recipients were HLA-DR matched (P < 0.0001). The etiology of this syndrome remains unknown. There was no evidence for graft vessel thrombosis. Because of some histological similarities, the usual causes of the hemolytic uremic syndrome, including bacterial and viral infections or cyclosporine arteriolopathy, were discussed. Acute vascular rejection was suspected, but the cross-match was negative on T lymphocytes in all cases and anti-HLA class I and II antibodies were not found to develop at the time of transplant dysfunction, except in 1 patient, in whom the detected anti-DR antibodies were not directed at the kidney donor. Anti-human umbilical vein endothelial cell antibodies, detected in an antibody-dependent cellular cytotoxicity assay, were present in 6 patients (of the 14 tested) at the onset of renal failure, but they were either absent (n = 3) or already present at the time of transplantation (n = 5) in the other 8 patients. Therefore, reliable arguments are lacking to conclude that this acute transplant dysfunction is an acute vascular rejection and its strong association with HLA matching has, as yet, no satisfactory explanation.

Polyclonal rabbit gamma globulins against a human cytotoxic CD4 T cell clone. II. Use in prevention of rejection in kidney transplantation: a pilot study.

Antiblast globulins (GAB) were prepared by immunization of rabbits with activated T lymphocytes (AT) derived from a rejected kidney allograft. AT consisted of a CD4+ (CD3+, CD2+ TCR alpha+ beta+) clone cytotoxic for HLA DR8-positive targets. The immunizing cells were adapted to industrial growth conditions by repetitive stimulations with an EBV-transformed line from the kidney donor and recombinant IL-2. In the pilot study, GAB (1.0-1.5 mg/kg/day) was given in 12-hr infusions, in association with prednisone (Pred) 1 mg/kg/day and azathioprine (Aza) 2 mg/kg/day, as prophylactic treatment of rejection in 12 kidney-transplanted patients during the first 2 weeks postgrafting. GAB dosage was further adapted according to the level of circulating E-rosette-forming T cells (ERFT). Cyclosporine A (8 mg/kg/day) was given at day 14 as a monotherapy after Pred and Aza were progressively tapered. No patient died, but one kidney was lost from surgical complication. No rejection occurred under GAB treatment; 41% of patients had at least one episode in the first 3 months and 16% from 3 to 9 months. GAB side effects were minor (skin rash: 2, low grade fever: 4) except for one acute serum sickness. Platelet and white blood cell counts were unchanged, but there was a significant decrease in hemoglobin during the 2 weeks of GAB infusions. Few infectious episodes occurred (3 bacterial, 2 viral). GAB monitoring showed a dramatic drop in T11+, T3+, T4+, and T8+ circulating T cells (less than 10% of normal values between days 3 and 14), whereas EFRT cells had a delayed and somewhat lower decrease (less than 10% after day 6 only). Consequently, mean GAB doses had to be raised to 1.3 mg/kg/day at day 4 and 1.6 at days 8 and 14. This pilot study suggests that this new bioreagent should be of major interest in the prophylaxis and treatment of rejection in allograft recipients. A controlled study is in progress.

DNA HLA-DR typing results of 4000 kidney transplants.

Recipients (4076) and donors (3325) of kidney transplants performed at 110 transplant centers were typed for HLA-DRB by the DNA RFLP method. The discrepancy rate of replicate samples distributed among 8 participating laboratories was a low 2.6%. The discrepancy rate between RFLP-DRB and serological HLA-DR typings was 25.0% for organ donors and 27.6% for kidney recipients. Discrepancy rates at the different transplant centers ranged from 9.7% to 86.7%. The discrepancies consisted of antigens being incorrectly interpreted by serology (16.8%), and of serological "blanks" turning out to be definable alleles by the DNA method (10.8%). The alleles that were mainly affected by discrepancies were DR1, DR8, DR10, DR12, DR13, DR14, DR16, DR17.2, and DR18.

Effects of MHC-encoded TAP1 and TAP2 gene polymorphism and matching on kidney graft rejection.

The products of TAP1 and TAP2 genes, recently mapped within the MHC class II region, are involved in antigen presentation by MHC class I molecules, especially in the transport of endogenous peptides. As for most MHC genes, a polymorphism has been described and the possibility that it could influence the recipient immune response by modulating antigen presentation in kidney transplantation has been tested. The aim of our study was to compare TAP1 and TAP2 gene polymorphism and matching in 53 couples of kidney donors and recipients without any rejection episodes and in 55 other couples who had experienced at least 2 acute cellular rejection episodes; 70 healthy individuals served as controls. Our results showed that allelic variant frequencies of TAP1 alleles (1A to 1C) and TAP2 alleles (2A to 2E), as assessed by amplification refractory mutation system-polymerase chain reaction, were similar among "rejection" and "no rejection" populations. Furthermore, there were no differences of TAP1 and/or TAP2 matching between donors and recipients in the 2 groups. In contrast, we showed that the recipients of the no rejection group were better matched with their corresponding donors for the HLA-DR genes than those of the rejection group. These results suggest that the currently described polymorphism in the limited coding region of TAP1 and TAP2 genes does not influence the incidence of kidney allograft rejection episodes and seems not to be a strong link to the adjacent DR/DQ subregion. Moreover, the observed increase frequency of TAP1B allele in the whole recipient's group as compared with controls (16.2% vs. 7.1% in the healthy individuals; P < 0.02) was not linked to the rejection occurrence but to the presence of glomerulonephritis as initial disease. Our study suggests that, in the clinical conditions tested, neither TAP polymorphism nor TAP matching influences the renal graft outcome.

Influence of HLA-A, B, and DR matching on the outcome of kidney transplant survival in preimmunized patients.

The outcome of 893 prospectively typed (HLA-A, B, and DR) and matched cadaveric kidney transplants--all first grafts, with patients being transfused before transplantation--was studied using actuarial survival methods. The effect of HLA-A, B and DR matching was only found to be significantly beneficial to graft survival in the group of 289 presensitized recipients: 70% and 43% graft survival at two years in the case of best-matched (4-6 HLA-A, B, and DR) identities versus mismatched (0 and 1 HLA-A, B, and DR) identities, respectively (P = 0.05). Although a cumulative effect of matching for antigens belonging to the 3 HLA-A, B, and DR series was observed among the group of preimmunized recipients, a trend arose in favor of the prominent role of the HLA-B alleles. No significant difference related to HLA matching was observed in the group of nonsensitized recipients. These results confirm previous observations and support efforts to give priority for matched kidneys to preimmunized patients.

Interaction of anti-HLA antibodies with pig xenoantigens.

BACKGROUND: Many patients with renal failure are condemned to long-term dialysis with little prospect of transplantation because they are highly sensitized with immunoglobulin G (IgG) directed against class I human leukocyte antigens (HLA) of virtually all donors. Xenotransplantation could represent an attractive solution providing their alloantibodies (alloAb) do not recognize porcine motifs. Hitherto there has been no in vivo demonstration of any cross-reactivity and the objective of this work was to investigate this problem using a technique of extracorporeal pig kidney perfusion as a model of clinical xenografting. METHODS: Pig kidneys were perfused ex vivo with plasma from both a group of highly sensitized patients and healthy individuals. Sequential plasma samples were analyzed for the titer of anti-Galalpha1-3Gal antibody (Ab) (major natural xenoreactive Ab) by enzyme-linked immunosorbent assay and anti-HLA class I Ab against a cell panel. At the end of perfusion, kidneys were perfused with a citric acid buffer to elute bound Ab. RESULTS: Galalpha1-3Gal Ab were shown to decrease rapidly in the plasma (in less than 10 min) and then reached a plateau. A fractional decrease in anti-HLA Ab was also found in some of the perfused plasma samples. Anti-Gal Ab were readily detected in all citric acid perfusates and anti-HLA Ab in 8 of 10. The HLA specificities of eluted Ab were mainly concordant with the originally designated specificities for each patient. CONCLUSION: Anti-HLA class I Ab presumably cross-react with pig class I homologues. However, some plasma samples did not cross-react, suggesting that negatively cross-matched pig kidneys could be identified in the pig population for xenotransplantation in these patients. Further studies are required to precisely describe these cross-reactivities and to understand their functional significance in xenotransplantation.

Crucial role of the third and fourth hypervariable regions of HLA-DPB1 allelic sequences in the mixed lymphocyte reaction.

Since HLA-DP mismatches are known to induce proliferative response in MLR I, we investigated the real impact of the different DP alleles and the possible role of one or several hypervariable regions of the DPB allelic sequences. Accordingly, we performed MLR I between HLA-A, B, DR, DQ, and Dw identical individuals DP oligotyped after DNA amplification. A total of 23 one-DP-mismatched healthy stimulator and responder cells displaying nine different DP specificities were thus evaluated in 52 MLRs I. This allowed us to analyze the impact of amino acid composition of each of the six hypervariable regions independently of the amino acid matching or mismatching in the five others. We show here that DP combinations sharing the same amino acid sequence in the third (C) and fourth (D) hypervariable regions are associated with a low proliferative response in vitro (p less than 0.01). These data imply that a perfect HLA-DP matching may not be requisite in selecting bone marrow donors. Indeed, the choice of donors may rely on determination of these particular mismatched HVRs between the DP alleles involved especially in GvHD direction. This policy including prospective DP oligotyping should be of great interest, especially when MLRs I are false negative or nonevaluable. It will enable a better definition of which DP mismatches are acceptable in BMT.

Susceptibility to alloimmunization to platelet HPA-1a antigen involves TAP1 polymorphism.

The alloimmunization against platelet HPA-1a antigen in mothers of thrombocytopenic neonates is strongly associated with HLA class II structures (DR3 and DR13) and especially with HLA-DR52a antigen (98% of the cases reported here). Because new genes have recently been mapped within the MHC class II region, we typed TAP1 and TAP2 gene polymorphisms by ARMS-PCR in order to characterize more effectively MHC genes involved in this alloimmunization. Our results showed that TAP1*0102 allele was significantly associated with NAIT only in the population of HLA-DR 13-DR52a-immunized women (50%) versus HLA-DR 13-DR52a controls (20%) (p < 0.05), and not in HLA-DR3-DR52a-immunized women versus HLA-DR3-DR52a controls. There is no linkage disequilibrium between TAP1*0102 and DRB1*13 alleles (delta = -0.0063) that could account for this result. The higher frequency of TAP1*0102 allele among HLA-DR 13-DR52a-immunized women suggests that HPA-1a antigen presentation and recognition may be influenced by nonclassic HLA class II gene polymorphisms, or that other linked but yet unknown genes could interfere.

Impact of the MHC-encoded HLA-DMA, DMB, and LMP2 gene polymorphisms on kidney graft outcome.

We previously studied the relationship between TAP1 and TAP2 gene polymorphism and compatibility in kidney graft outcome and reported that the currently described TAP1 and TAP2 gene polymorphisms did not influence the incidence of acute rejection episodes. In this study, we report on the effect of polymorphism and matching of HLA-DMA, -DMB, and LMP2 genes on kidney graft outcome. This study was performed on 102 selected kidney recipients who experienced two or more acute rejection episodes (rejection group) during follow up and who were compared to a group of 150 patients who never had rejection (non rejection group). Although a significant effect of HLA-DR matching was observed between these two groups, our data suggest that matching for all the new genes located in the HLA class II region (TAP1, TAP2, LMP2, HLA-DMA and -DMB) does not influence the kidney graft outcome. However, a significant increase (pc < 0.05) of DMA*0102 allele was observed in the recipients of the rejection group as compared to those of the non rejection group. This effect was not due to a linkage disequilibrium between DMA and HLA-DR loci and suggests that this specific HLA-DMA allele could play a role in the indirect pathway of class II presentation of donor antigens.

HLA-DRw52a is involved in alloimmunization against PL-A1 antigen.

The alloimmunization against the platelet PL-A1 antigen is strongly associated with a HLA class II structure in mothers of thrombocytopenic neonates. Most of the immunized women have first been shown to possess the DR3 specificity and subsequently the DRw52 allele. The 18 immunized mothers studied here by restriction fragment length polymorphism analysis had the DRw52a specificity at the DRB3 locus whatever their HLA-DRB1 gene product. This finding strongly suggests that the DRB3 chain is directly involved in the presentation of the PL-A1 antigen to the specific T cell. In addition, the similarities between DR3 and DRw52 structures due to a hypothetical gene conversion event should be considered in order to understand the high frequency of DR3 among the DRw52a-responding women. Alternatively, the high frequency of DR3 among the DRw52-responding mothers might be due to the high responder status associated with the former specificity.

Influence of HLA-DP mismatches on primary MLR responses in unrelated HLA-A, B, DR, DQ, Dw identical pairs in allogeneic bone marrow transplantation.

The success of bone marrow transplantation relies on class I and class II HLA identity between donor and recipient but until now the impact of DP mismatches on primary mixed lymphocyte reaction (MLR I) responses has not yet been clearly established. HLA-DP typing has been performed by a RFLP method on 10 patients and their 22 selected potential donors (HLA-A, B, DR, DQ, Dw identical according to serological, RFLP and oligotyping methods). HLA-DP-matching was then correlated with MLR I responses obtained (1) in 22 HLA-A, B, DR, DQ, Dw identical donor-recipient pairs (patient series) and (2) in 30 HLA-A, B, DR, DQ, Dw identical donors pairs used as the control series. We showed that MLR I responses involving patients cells were always lower than those involving control cells (p less than 0.02 in case of two DP-mismatches and p less than 0.001 in the case of one DP-mismatch). Moreover, in the control series two or one DP mismatches influenced MLR I responses in 91% and 77% of the cases respectively; however, there was a greater scatter of response values which could be explained by the degree of sequence homology between the DP mismatched alleles. In cases with no DP mismatch, no proliferative response was observed. Overall, these results suggest that the conventional technical conditions of MLR I do not allow detection of mismatches other than the well known HLA specificities.

No evidence of HTLV-I infection in French patients with multiple sclerosis using the polymerase chain reaction.

The polymerase chain reaction (PCR), using three primer pairs in the pol, tax, and env regions of the HTLV-I genome, was unable to detect HTLV-I in the blood samples of 54 caucasian subjects with multiple sclerosis who were seronegative for HTLV-I/II. Seventeen HTLV-I/II seropositive (by ELISA and Western blot) subjects used as positive controls were positive with the three primer pairs. The PCR was negative in 47 healthy HTLV-I/II seronegative (by ELISA) subjects at low risk of HTLV-I infection used as negative controls. These results suggest that there is no association between the occurrence of HTLV-I sequences and the development of multiple sclerosis.